In the field of biomedicine, there is a significant need for bioassays useful to determine the activity of body fluid components, particularly enzymes such as proteases, on their respective target substrates. Although chromogenic or fluorogenic substrates have been used in the past to assay the activity of enzymes, there is a need for a method or assay which is rapid, efficient, inexpensive, and highly sensitive.
For example, an illustration of the usefulness of such assays is the measurement of components involved in fibrinogen polymerization, fibrinolysis, or fibrinogenolysis. Fibrinogen is the key structural protein in blood clot formation. It is a fibrous protein having three pairs of polypeptide chains held together by disulfide bonds. In fibrinogen polymerization, as illustrated in FIG. 1A, fibrinogen is a substrate for thrombin proteolytic activity. Thrombin catalyzes the release of small peptides, fibrinopeptides A and B, from the chains of fibrinogen. The removal of the fibrinopeptides from the fibrinogen substrate results in molecules which polymerize spontaneously into fibers in forming fibrin. After serving its role as the protein matrix of a blood clot, and in tissue repair, fibrin is then removed or degraded in the process of fibrinolysis.
As illustrated in FIG. 1B, the inactive precursor, plasminogen, is converted into the active proteolytic enzyme, plasmin. In fibrinolysis, plasmin catalyzes the fibrin substrate into soluble degradation products. Further, since plasmin is enzymatically active against fibrinogen, fibrinogenolysis is a process by which fibrinogen substrate is degraded into soluble products.
Measurement of the various enzymes, inhibitors, activators, and protein substrates involved in clot formation, inhibition of clot formation, or dissolution of clots (thrombolysis) has medical applications. For example, thrombosis is an important cause of human illness and death. Thrombolytic agents are increasingly used in pharmacological prevention and/or dissolution of formed thrombi such as in acute myocardial infarction.
Various methods for assaying various blood factors involved with the formation, inhibition, or degradation of clots have been described previously. Typically, these bioassays involve chromogenic assay techniques requiring enzyme labelling of the substrate, addition of the component(s) for which activity is to be measured, and subsequent detection of the amount of color formed. Similar bioassays have been described without the use of chromogens, but instead measure turbidity once the reaction has taken place. Disadvantages of these types of assays, particularly for the measurement of components which are normally in low concentrations in body fluid, include: susceptibility to diffusion artifacts; relative time consumption to perform the assay and obtain the results; the concentration of substrate that is required for the assay; lack of kinetic information of the process being measured; and lack of sensitivity of detection to the picomolar or nanomolar level. Thus, for applications such as monitoring thrombolytic agent therapy in a clinical setting, previously described methods have been found to be time-consuming and insufficiently sensitive.
Therefore, there is interest and need in providing assays which are rapid, highly sensitive, provide measurements of body fluid components which reliably reflect in vivo activity, and which can be used to detect and quantitate a wide variety of analytes including enzymes, enzyme inhibitors, and enzyme activators.